Enhanced Polar Auxin Transport in Tomato Polycotyledon Mutant Seems to be Related to Glutathione Levels

Glutathione-dependent root growth in Arabidopsis is linked to polar auxin transport (PAT). Arabidopsis mutants with reduced glutathione (GSH) levels also show reduced PAT. To gain an insight into the relationship between PAT and GSH level, we analyzed tomato polycotyledon mutant, pct1-2, which has enhanced PAT. Microarray analysis of gene expression in pct1-2 mutant revealed underexpression of several genes related to glutamate and glutathione metabolism. In consonance with microarray analysis, enzymatic as well as in vivo assay revealed higher glutathione levels in the early phase of pct1-2 seedling growth than WT. The inhibition of auxin transport by 2,3,5-triiodobenzoic acid (TIBA) reduced both GSH level and PIN1 expression in pct1-2 root tips. The reduction of in vivo GSH accumulation in pct1-2 root tips by buthionine sulfoximine (BSO) stimulated elongation of the short root of pct1-2 mutant akin to TIBA. The rescue of the short root phenotype of the pct1-2 mutant was restricted to TIBA and BSO. The other auxin transport inhibitors 1-N-naphthylphthalamic acid (NPA), 2-[4-(diethylamino)-2-hydroxybenzoyl] benzoic acid (BUM), 3-chloro-4-hydroxyphenylacetic acid (CHPAA), brefeldin and gravacin inhibited root elongation in both WT and pct1-2 mutant. Our results indicate a relationship between PAT and GSH levels in tomato akin to Arabidopsis. Our work also highlights that TIBA rescues the short root phenotype of the pct1-2 mutant by acting on a PAT component distinct from the site of action of other PAT inhibitors.

     

Phosphatidylinositol‐specific phospholipase C2 functions in auxin‐modulated root development

Nine phosphatidylinositol‐specific phospholipases C (PLCs) have been identified in the Arabidopsis genome; among the importance of PLC2 in reproductive development is significant. However, the role of PLC2 in vegetative development such as in root growth is elusive. Here, we report that plc2 mutants displayed multiple auxin‐defective phenotypes in root development, including short primary root, impaired root gravitropism, and inhibited root hair growth. The DR5:GUS expression and the endogenous indole‐3‐acetic acid (IAA) content, as well as the responses of a set of auxin‐related genes to exogenous IAA treatment, were all decreased in plc2 seedlings, suggesting the influence of PLC2 on auxin accumulation and signalling. The root elongation of plc2 mutants was less sensitive to the high concentration of exogenous auxins, and the application of 1‐naphthaleneacetic acid or the auxin transport inhibitor N‐1‐naphthylphthalamic acid could rescue the root hair growth of plc2 mutants. In addition, the PIN2 polarity and cycling in plc2 root epidermis cells were altered. These results demonstrate a critical role of PLC2 in auxin‐mediated root development in Arabidopsis, in which PLC2 influences the polar distribution of PIN2.     

Phytochrome B control of total leaf area and stomatal density affects drought tolerance in rice

We report that phytochrome B (phyB) mutants exhibit improved drought tolerance compared to wild type (WT) rice (Oryza sativa L. cv. Nipponbare). To understand the underlying mechanism by which phyB regulates drought tolerance, we analyzed root growth and water loss from the leaves of phyB mutants. The root system showed no significant difference between the phyB mutants and WT, suggesting that improved drought tolerance has little relation to root growth. However, phyB mutants exhibited reduced total leaf area per plant, which was probably due to a reduction in the total number of cells per leaf caused by enhanced expression of Orysa;KRP1 and Orysa;KRP4 (encoding inhibitors of cyclin-dependent kinase complex activity) in the phyB mutants. In addition, the developed leaves of phyB mutants displayed larger epidermal cells than WT leaves, resulting in reduced stomatal density. phyB deficiency promoted the expression of both putative ERECTA family genes and EXPANSIN family genes involved in cell expansion in leaves, thus causing greater epidermal cell expansion in the phyB mutants. Reduced stomatal density resulted in reduced transpiration per unit leaf area in the phyB mutants. Considering all these findings, we propose that phyB deficiency causes both reduced total leaf area and reduced transpiration per unit leaf area, which explains the reduced water loss and improved drought tolerance of phyB mutants.     

Impaired phytochrome-mediated shade-avoidance responses in the aurea mutant of tomato

Internode extension-growth responses to neighbouring plants and to red to far-red ratios (R:FR) were investigated in wild-type (WT) and aurea (au)-mutant seedlings of tomato grown under natural radiation. The genomic location of the au mutant is not known, but one of its consequences is the reduced phytochrome level. In WT seedlings, internode growth was promoted by the presence of non-shading neighbours reflecting far-red light (FR), the shade of a tall canopy, FR provided as a supplement during the photoperiod, and FR pulses either provided at the end of the day or delayed into the dark period. Supplementary FR during the photoperiod also promoted growth in herbicide-treated partially bleached WT seedlings. The au mutant showed higher background extension-growth rates, but only responded to the most severe treatments: deep shade light and very low R:FR at the end of the day, i.e. au-mutant seedlings were less sensitive than WT seedlings to R:FR signals. Wild-type seedlings were transferred from the glasshouse to a growth room and exposed to white light with two levels of phytochrome-absorbable radiation but similar phytochrome photoequilibria and radiation for photosynthesis. The plants exposed to the lowest level showed a transient increase of internode extension growth rate and a simultaneous reduction of response to FR pulses, i.e. reproduced some of the features of au-mutant seedlings. Phytochrome itself could set the degree of response to Pfr during neighbour detection.     

The auxin transporter,OsAUX1, is involved in primary root and root hair elongation and in Cd stress responses in rice (Oryza sativa L.)

Auxin and cadmium (Cd) stress play critical roles during root development. There are only a few reports on the mechanisms by which Cd stress influences auxin homeostasis and affects primary root (PR) and lateral root (LR) development, and almost nothing is known about how auxin and Cd interfere with root hair (RH) development. Here, we characterize rice osaux1 mutants that have a longer PR and shorter RHs in hydroponic culture, and that are more sensitive to Cd stress compared to wild‐type (Dongjin). OsAUX1 expression in root hair cells is different from that of its paralogous gene, AtAUX1, which is expressed in non‐hair cells. However, OsAUX1, like AtAUX1, localizes at the plasma membrane and appears to function as an auxin tranporter. Decreased auxin distribution and contents in the osaux1 mutant result in reduction of OsCyCB1;1 expression and shortened PRs, LRs and RHs under Cd stress, but may be rescued by treatment with the membrane‐permeable auxin 1‐naphthalene acetic acid. Treatment with the auxin transport inhibitors 1‐naphthoxyacetic acid and N‐1‐naphthylphthalamic acid increased the Cd sensitivity of WT rice. Cd contents in the osaux1 mutant were not altered, but reactive oxygen species‐mediated damage was enhanced, further increasing the sensitivity of the osaux1 mutant to Cd stress. Taken together, our results indicate that OsAUX1 plays an important role in root development and in responses to Cd stress.     

The auxin influx carrier,OsAUX3, regulates rice root development and responses to aluminium stress

In rice, there are five members of the auxin carrier AUXIN1/LIKE AUX1 family; however, the biological functions of the other four members besides OsAUX1 remain unknown. Here, by using CRISPR/Cas9, we constructed two independent OsAUX3 knock‐down lines, osaux3‐1 and osaux3‐2, in wild‐type rice, Hwayoung (WT/HY) and Dongjin (WT/DJ). osaux3‐1 and osaux3‐2 have shorter primary roots (PRs), decreased lateral root (LR) density, and longer root hairs (RHs) compared with their WT. OsAUX3 expression in PRs, LRs, and RHs further supports that OsAUX3 plays a critical role in the regulation of root development. OsAUX3 locates at the plasma membrane and functions as an auxin influx carrier affecting acropetal auxin transport. OsAUX3 is up‐regulated in the root apex under aluminium (Al) stress, and osaux3‐2 is insensitive to Al treatments. Furthermore, 1‐naphthylacetic acid accented the sensitivity of WT/DJ and osaux3‐2 to respond to Al stress. Auxin concentrations, Al contents, and Al‐induced reactive oxygen species‐mediated damage in osaux3‐2 under Al stress are lower than in WT, indicating that OsAUX3 is involved in Al‐induced inhibition of root growth. This study uncovers a novel pathway alleviating Al‐induced oxidative damage by inhibition of acropetal auxin transport and provides a new option for engineering Al‐tolerant rice species.     

A phytochrome-mediated alteration from stem to rosette growth on chicory root explants in vitro

Chicory root explants (Cichorium intybus L. var. foliosum) of two cultivars, taken before and after hydroponic forcing, were cultured in vitro in complete darkness supplemented with red and far-red light treatments. Using 5 min red light per day, the strong stem elongation occurring in complete darkness was converted to rosette formation. This reaction was reversed to stem elongation (accompanied by leaf formation) adding 15 min far-red light after the red light. Fifteen min far-red light per day alone caused the same reaction as 5 min red/15 min far-red light. Far-red light followed by red light caused rosette formation. In stems, formed under complete darkness in vitro, the presence of phytochrome was shown. No phytochrome was detected in the root fragment itself.Abbreviations R red light - FR far-red light - GA gibberellinic acid - A absorbance - FW fresh weight     

Auxin and its transport play a role in plant tolerance to arsenite‐induced oxidative stress in Arabidopsis thaliana

The role of auxin in plant development is well known; however, its possible function in root response to abiotic stress is poorly understood. In this study, we demonstrate a novel role of auxin transport in plant tolerance to oxidative stress caused by arsenite. Plant response to arsenite [As(III)] was evaluated by measuring root growth and markers for stress on seedlings treated with control or As(III)‐containing medium. Auxin transporter mutants aux1, pin1 and pin2 were significantly more sensitive to As(III) than the wild type (WT). Auxin transport inhibitors significantly reduced plant tolerance to As(III) in the WT, while exogenous supply of indole‐3‐acetic acid improved As(III) tolerance of aux1 and not that of WT. Uptake assays using H³‐IAA showed As(III) affected auxin transport in WT roots. As(III) increased the levels of H₂O₂ in WT but not in aux1, suggesting a positive role for auxin transport through AUX1 on plant tolerance to As(III) stress via reactive oxygen species (ROS)‐mediated signalling. Compared to the WT, the mutant aux1 was significantly more sensitive to high‐temperature stress and salinity, also suggesting auxin transport influences a common element shared by plant tolerance to arsenite, salinity and high‐temperature stress.     

Temporal root responses in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> L. to chromate reveal structural and regulatory mechanisms involving the SOLITARY ROOT/IAA14 repressor for maintenance of identity meristem genes

The Arabidopsis root system is modified in response to stress generated by high concentrations of nonessential ions such as chromate [Cr(VI)]. In this work, the distribution of auxin and its transporters PIN1 and PIN7, as well as the expression of genes that maintain the identity of the root meristem, were analyzed in Arabidopsis thaliana wild-type (WT) seedlings and in a mutant affected in the SOLITARY ROOT (SLR1/IAA14) locus, which is required for root response to Cr(VI). We show that primary root inhibition, auxin transporter levels, and expression of meristem identity genes were maintained in the slr-1 mutants but not in WT plants in response to Cr(VI) in a time- and concentration-dependent manner. Notably, the outermost single cell layer of the lateral root cap, which normally dies and tends to peel off, remains viable and increases in size following exposure of WT plants, but not slr-1 mutants, to Cr(VI). Our results suggest that (1) the primary root tip senses Cr(VI), (2) the external lateral root cap may play a protective role during Cr(VI) exposure, and (3) Cr(VI) impacts cell division in root meristems via auxin redistribution and SLR1/IAA14 function, influencing the expression of root meristem genes.     

Two homologous ATP-binding cassette transporter proteins, AtMDR1 and AtPGP1, regulate Arabidopsis photomorphogenesis and root development by mediating polar auxin transport

Light and auxin control many aspects of plant growth and development in an overlapping manner. We report here functional characterization of two closely related ABC (ATP-binding cassette) transporter genes, AtMDR1 and AtPGP1, in light and auxin responses. We showed that loss-of-function atmdr1 and atpgp1 mutants display hypersensitivity to far-red, red, and blue-light inhibition of hypocotyl elongation, reduced chlorophyll and anthocyanin accumulation, and abnormal expression of several light-responsive genes, including CAB3, RBCS, CHS, and PORA, under both darkness and far-red light conditions. In addition, we showed that the atmdr1-100 and atmdr1-100/atpgp1-100 mutants are defective in multiple aspects of root development, including increased root-growth sensitivity to 1-naphthalene acetic acid (1-NAA), and decreased sensitivity to naphthylphthalamic acid (NPA)-mediated inhibition of root elongation. Consistent with the proposed role of AtMDR1 in basipetal auxin transport, we found that expression of the auxin responsive DR5::GUS reporter gene in the central elongation zone is significantly reduced in the atmdr1-100 mutant roots treated with 1-NAA at the root tips, compared to similarly treated wild-type plants. Moreover, atmdr1-100, atpgp1-100, and their double mutants produced fewer lateral roots, in the presence or absence of 1-NAA or NPA. The atmdr1-100 and atmdr1-100/atpgp1-100 mutants also displayed enhanced root gravitropism. Genetic-epistasis analysis revealed that mutations in phyA largely suppress the randomized-hypocotyl growth and the short-hypocotyl phenotype of the atmdr1-100 mutants under far-red light, suggesting that phyA acts downstream of AtMDR1. Together, our results suggest that AtMDR1 and AtPGP1 regulate Arabidopsis (Arabidopsis thaliana) photomorphogenesis and multiple aspects of root development by mediating polar auxin transport.     

Cadmium interferes with maintenance of auxin homeostasis in Arabidopsis seedlings

Auxin and its homeostasis play key roles in many aspects of plant growth and development. Cadmium (Cd) is a phytotoxic heavy metal and its inhibitory effects on plant growth and development have been extensively studied. However, the underlying molecular mechanism of the effects of Cd stress on auxin homeostasis is still unclear. In the present study, we found that the root elongation, shoot weight, hypocotyl length and chlorophyll content in wild-type (WT) Arabidopsis seedlings were significantly reduced after exposure to Cd stress. However, the lateral root (LR) formation was markedly promoted by Cd stress. The level and distribution of auxin were both greatly altered in primary root tips and cotyledons of Cd-treated plants. The results also showed that after Cd treatment, the IAA content was significantly decreased, which was accompanied by increases in the activity of the IAA oxidase and alteration in the expression of several putative auxin biosynthetic and catabolic genes. Application of the auxin transport inhibitor, 1-naphthylphthalamic acid (NPA) and 1-naphthoxyacetic acid (1-NOA), reversed the effects of Cd on LR formation. Additionally, there was less promotion of LR formation by Cd treatment in aux1-7 and pin2 mutants than that in the WT. Meanwhile, Cd stress also altered the expression of PINs and AUX1 in Arabidopsis roots, implying that the auxin transport pathway is required for Cd-modulated LR development. Taken together, these findings suggest that Cd stress disturbs auxin homeostasis through affecting auxin level, distribution, metabolism, and transport in Arabidopsis seedling.     

Phytochrome regulation of greening in wild type and long-hypocotyl mutants ofArabidopsis thaliana

A brief pulse of red light (R) given to darkgrown seedlings ofArabidopsis thaliana (L.) Heyn. potentiates rapid synthesis of chlorophyll upon transfer to continuous white light. The time course for potentiation of rapid greening shows that a R pulse in the LF (low fluence) range has maximal effect within a few hours, and that there is a small VLF (very low fluence) component as well. Partial reversal of the effect of R by far-red light (FR) indicates that the pulse acts through phytochrome. As it does in the wild-type (WT), a pulse of R accelerates greening of long-hypocotyl (hy) mutants. The extent of induction by the R pulse was about the same in the WT and in allhy mutants studied. Reversibility by FR was greatly decreased in thehy-1 andhy-2 strains. It is possible that these mutants contain a species of phytochrome with defective phototransformation kinetics. If there is such a defective phytochrome species, it nevertheless appears to be active in the potentiation of rapid greening. Dedicated to Professor Hans Mohr on the occasion of his 60th birthday     

A strigolactone signal is required for adventitious root formation in rice

Background and Aims Strigolactones (SLs) and their derivatives are plant hormones that have recently been identified as regulating root development. This study examines whether SLs play a role in mediating production of adventious roots (ARs) in rice (Oryza sativa), and also investigates possible interactions between SLs and auxin.Methods Wild-type (WT), SL-deficient (d10) and SL-insensitive (d3) rice mutants were used to investigate AR development in an auxin-distribution experiment that considered DR5::GUS activity, [³H] indole-3-acetic acid (IAA) transport, and associated expression of auxin transporter genes. The effects of exogenous application of GR24 (a synthetic SL analogue), NAA (α-naphthylacetic acid, exogenous auxin) and NPA (N-1-naphthylphalamic acid, a polar auxin transport inhibitor) on rice AR development in seedlings were investigated.Key Results The rice d mutants with impaired SL biosynthesis and signalling exhibited reduced AR production compared with the WT. Application of GR24 increased the number of ARs and average AR number per tiller in d10, but not in d3. These results indicate that rice AR production is positively regulated by SLs. Higher endogenous IAA concentration, stronger expression of DR5::GUS and higher [³H] IAA activity were found in the d mutants. Exogenous GR24 application decreased the expression of DR5::GUS, probably indicating that SLs modulate AR formation by inhibiting polar auxin transport. The WT and the d10 and d3 mutants had similar expression of DR5::GUS regardless of exogenous application of NAA or NPA; however, AR number was greater in the WT than in the d mutants.Conclusions The results suggest that AR formation is positively regulated by SLs via the D3 response pathway. The positive effect of NAA application and the opposite effect of NPA application on AR number of WT plants also suggests the importance of auxin for AR formation, but the interaction between auxin and SLs is complex.     

Auxin–cytokinin and auxin–gibberellin interactions during morphogenesis of the compound leaves of pea (<Emphasis Type="Italic">Pisum sativum</Emphasis>)

A number of mutations that alter the form of the compound leaf in pea (Pisum sativum) has proven useful in elucidating the role that auxin might play in pea leaf development. The goals of this study were to determine if auxin application can rescue any of the pea leaf mutants and if gibberellic acid (GA) plays a role in leaf morphogenesis in pea. A tissue culture system was used to determine the effects of various auxins, GA or a GA biosynethesis inhibitor (paclobutrazol) on leaf development. The GA mutant, nana1 (na1) was analyzed. The uni-tac mutant was rescued by auxin and GA and rescue involved both a conversion of the terminal leaflet into a tendril and an addition of a pair of lateral tendrils. This rescue required the presence of cytokinin. The auxins tested varied in their effectiveness, although methyl-IAA worked best. The terminal tendrils of wildtype plantlets grown on paclobutrazol were converted into leaflets, stubs or were aborted. The number of lateral pinna pairs produced was reduced and leaf initiation was impaired. These abnormalities resembled those caused by auxin transport inhibitors and phenocopy the uni mutants. The na1 mutant shared some morphological features with the uni mutants; including, flowering late and producing leaves with fewer lateral pinna pairs. These results show that both auxin and GA play similar and significant roles in pea leaf development. Pea leaf morphogenesis might involve auxin regulation of GA biosynthesis and GA regulation of Uni expression.     

OsPIN2, which encodes a member of the auxin efflux carrier proteins,is involved in root elongation growth and lateral root formation patterns via the regulation of auxin distribution in rice

Auxin flow is important for different root developmental processes such as root formation, emergence, elongation and gravitropism. However, the detailed information about the mechanisms regulating the auxin flow is less well understood in rice. We characterized the auxin transport‐related mutants, Ospin‐formed2‐1 (Ospin2‐1) and Ospin2‐2, which exhibited curly root phenotypes and altered lateral root formation patterns in rice. The OsPIN2 gene encodes a member of the auxin efflux carrier proteins that possibly regulates the basipetal auxin flow from the root tip toward the root elongation zone. According to DR5‐driven GUS expression, there is an asymmetric auxin distribution in the mutants that corresponded with the asymmetric cell elongation pattern in the mutant root tip. Auxin transport inhibitor, N‐1‐naphthylphthalamic acid and Ospin2‐1 Osiaa13 double mutant rescued the curly root phenotype indicating that this phenotype results from a defect in proper auxin distribution. The typical curly root phenotype was not observed when Ospin2‐1 was grown in distilled water as an alternative to tap water, although higher auxin levels were found at the root tip region of the mutant than that of the wild‐type. Therefore, the lateral root formation zone in the mutant was shifted basipetally compared with the wild‐type. These results reflect that an altered auxin flow in the root tip region is responsible for root elongation growth and lateral root formation patterns in rice.